Integrated genomic analysis reveals aberrations in WNT signaling in germ cell tumors of childhood and adolescence

Germ cell tumors (GCTs) are neoplasms of the testis, ovary and extragonadal sites that occur in infants, children, adolescents and adults. Post-pubertal (type II) malignant GCTs may present as seminoma, non-seminoma or mixed histologies. In contrast, pre-pubertal (type I) GCTs are limited to (benign) teratoma and (malignant) yolk sac tumor (YST). Epidemiologic and molecular data have shown that pre- and post-pubertal GCTs arise by distinct mechanisms. Dedicated studies of the genomic landscape of type I and II GCT in children and adolescents are lacking. Here we present an integrated genomic analysis of extracranial GCTs across the age spectrum from 0–24 years. Activation of the WNT pathway by somatic mutation, copy-number alteration, and differential promoter methylation is a prominent feature of GCTs in children, adolescents and young adults, and is associated with poor clinical outcomes. Significantly, we find that small molecule WNT inhibitors can suppress GCT cells both in vitro and in vivo. These results highlight the importance of WNT pathway signaling in GCTs across all ages and provide a foundation for future efforts to develop targeted therapies for these cancers.


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Data collection
No software was used Data analysis For whole-exome sequencing data, raw reads was mapped to human reference genome (hg19) using BWA (version bwa-0.7.17). Local realignment and base quality recalibration were performed using default parameters by GATK pipeline (version 3.7). Matched tumornormal BAM files were used as input for VarScan software (version 2.4.0) to identify somatic single-nucleotide variants (SNVs) and smallscale insertion/deletions (INDELs). For RNA sequencing data, The generated FASTQ files were aligned by Bowtie2 (version 2.3) and TopHat2 (version 2.1.1). Cufflinks (version 2.2.1) was used to assemble and estimate the relative abundances of transcripts at the gene and transcript level. SNP array data was processed by Nexus Copy Number Discovery 7.0 software (BioDiscovery, Inc.). Methylation array data was processed by methylumi package (version 3.7) and IMA package (version 3.1.2). Image analysis was performed with ImageJ (version 1.53m).
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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Genomic sequencing data of germ cell tumor samples in this study are deposited to dbGaP (https://www.ncbi.nlm.nih.gov/gap/) with Accession Number phs002009.v1.p1. Tumor methylation data are deposited in the Gene Expression Omnibus (GEO) repository under Accession GSE183798. The following databases were used in the analysis: 1000 Genome Project database (https://www.internationalgenome.org); National Heart, Lung, and Blood Institute (NHLBI) Exome Variant

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Sample size
Genomic profiling of tumors was performed to determine the landscape of somatic mutations in pediatric germ cell tumors. No sample size calculation was performed. Malignant germ cell tumor is a rare diagnosis and represents less than 2% of all childhood cancers. The cases profiled represent all cases for which informed consent had been obtained, deidentified clinical annotation was available, and a specimen of suitable sample quality was available in the biospecimen repositories of UT Southwestern Medical Center, Boston Children's Hospital, the Erasmus University Medical Center and the Hospital Sant Joan de Déu, Barcelona, Spain as well as the Children's Oncology Group Biopathology Center, since 2009. This study is -to our knowledge -the largest cohort of pediatric germ cell tumors described to date. For experiments involving animal models, a power calculation was performed using ClinCalc (https://clincalc.com/stats/samplesize.aspx) and sample sizes were chosen to provide 80% to detect a difference in means of 30% with alpha = 0.05.

Replication
Cell growth experiments were performed using two different small-molecule WNT inhibitors with different mechanisms of action. 4 indepdendent cell lines were used and all cell lines were validated by STR genotyping and confirmed to be free of mycoplasma contamination. Cell growth studies were performed in triplicate. All experiments for cell growth, immunostaining and in vivo drug treatment studies were replicated twice and all replicates were successful.
Randomization Zebrafish in vivo studies: Animals with tumors were randomly assigned to vehicle control or drug treatment groups. For analysis of human tumor specimens, for some analyses tumors were classified as Type I or Type II according to established criteria as described in the manuscript. Randomization does not apply as there were no within-group comparisons.

Blinding
All measurements including cell line drug exposures, GFP fluorescence images and gH2AX immunostains in drug-treated animals were quantified by personnel blinded to the treatment arm. For bioinformatic analyses, all samples were deidentified at the source and assigned a study number; bioinformatics analysis was performed without reference to associated clinical data except for analyses that compared Type I and Type II tumors.

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